The field of transgenics was initially developed to understand the action of a single gene in the context of the whole animal and the phenomena of gene activation, expression, and interaction. This technology has also been used to produce models for various diseases in humans and other animals and is amongst the most powerful tools available for the study of genetics, and the understanding of genetic mechanisms and function. From an economic perspective, the use of transgenic technology for the production of specific proteins or other substances of pharmaceutical interest (Gordon et al., Biotechnology 5: 1183-1187 (1987); Wilmut et al., Theriogenology 33: 113-123 (1990)) offers significant advantages over more conventional methods of protein production by gene expression.
Heterologous nucleic acids have been engineered so that an expressed protein may be joined to a protein or peptide that will allow secretion of the transgenic expression product into milk or urine, from which the protein may then be recovered. These procedures have had limited success and may require lactating animals, with the attendant costs of maintaining individual animals or herds of large species, including cows, sheep, or goats.
Historically, transgenic animals have been produced almost exclusively by microinjection of the fertilized egg. The pronuclei of fertilized eggs are microinjected in vitro with foreign, i.e., xenogeneic or allogeneic, heterologous DNA or hybrid DNA molecules. The microinjected fertilized eggs are then transferred to the genital tract of a pseudopregnant female (See e.g., Krimpenfort et al., in U.S. Pat. No. 5,175,384).
One system that holds potential is the avian reproductive system. The production of an avian egg begins with formation of a large yolk in the ovary of the hen. The unfertilized oocyte or ovum is positioned on top of the yolk sac. After ovulation, the ovum passes into the infindibulum of the oviduct where it is fertilized, if sperm are present, and then moves into the magnum of the oviduct which is lined with tubular gland cells. These cells secrete the egg-white proteins, including ovalbumin, ovomucoid, ovoinhibitor, conalbumin, ovomucin and lysozyme, into the lumen of the magnum where they are deposited onto the avian embryo and yolk.
The hen oviduct offers outstanding potential as a protein bioreactor because of the high levels of protein production, the promise of proper folding and post-translation modification of the target protein, the ease of product recovery, and the shorter developmental period of chickens compared to other potential animal species. As a result, efforts have been made to create transgenic chickens expressing heterologous proteins in the oviduct.
Chicken oviduct cells, when stimulated by steroid hormones during egg-laying, secrete three principal polypeptides, ovalbumin, ovomucoid and lysozyme (Tsai et al., Biochemistry 17: 5773-5779 (1978)). The mRNA transcript encoding ovalbumin constitutes about 50% of the total mRNA of these cells. Ovomucoid and lysozyme mRNAs contribute about 6.6% and 3.4% respectively of the total mRNA of the steroid stimulated cells. (Hynes et al., Cell 11:923-932 (1977)).
Detailed restriction enzyme analysis of fragments of chicken genomic DNA have shown that the ovomucoid-encoding sequence includes seven intronic sequences (Lindenmaier et al., Nuc. Acid Res. 7;1221-1232 (1979); Catterall et al., Nature 278: 323-327 (1979); Lai et al., Cell 18:829-842 (1979)). Short stretches of the 5′ flanking region of the ovomucoid gene have been sequenced (Lai et al., Cell 18: 829-842 (1979); Genbank Accession No. J00897), but extending only 579 bases upstream of the recognized transcription start site. The 5′ flanking region of the ovomucoid gene has been isolated (Catterall et al., Nature 278: 323-327 (1979); Lai et al., Cell 18: 829-842 (1979)), but not generally characterized beyond low-resolution restriction site mapping. Scott et al. in Biochemistry 26: 6831-6840 (1987) identified a CR1-like region within the 10 kb chicken genomic DNA located between the ovoinhibitor-encoding region and the downstream ovomucoid gene. The ovoinhibitor-encoding cDNA and the attached 3′-untranslated region, which extends into the 10 kb ovoinhibitor-ovomucoid region, were also sequenced (Scott et al. J. Biol. Chem. 262: 5899-5907 (1987)).
The chicken ovomucoid gene, therefore, is highly expressed in the tubular glands of the mature hen oviduct and represents a suitable candidate for an efficient promoter for heterologous protein production in transgenic animals, especially animals. The regulatory region of the ovomucoid locus extends over a nucleic acid region of about 10 kb of DNA 5′ upstream of the transcription start site, and comprises at least one recognized element, the CR1.